It can be produced in nuclear research reactors, irradiating isotopically-enriched 168Er2O3. This path, however, is not suitable for receptor-targeted radionuclide therapy, where high specific molar activities are required. In this study, an electromagnetic isotope separation technique was applied after neutron irradiation to boost the specific activity by separating 169Er from 168Er targets. The separation efficiency increased up to 0.5% using resonant laser ionization. A subsequent chemical purification process was developed as well as activity standardization of the radionuclidically pure 169Er. The quality of the 169Er product permitted radiolabeling and pre-clinical studies. A preliminary in vitro experiment was accomplished, using a 169Er-PSMA-617, to show the potential of 169Er to reduce tumor cell viability.The β Radiolanthanides are of particular interest in the field of nuclear medicine, offering attractive decay properties for both diagnosis and therapy –6. One o−-emitting 177Lu is used on a routine basis in clinics for targeted radionuclide therapy. The Center of Radiopharmaceutical Sciences (CRS) at Paul Scherrer Institute (PSI) has recently introduced 161Tb as a potentially better alternative to 177Lu due to its co-emission of conversion and Auger electrons  with the pure Auger-electron emitter 165Er can, thus, represent an ideal model system to evaluate the additive therapeutic effect of Auger electrons on targeted β− therapy.Currently, the βlectrons . There i169Er  have promising decay properties toward radionuclide therapy of metastasized cancer diseases  values than the currently clinically-used radiolanthanide 177Lu   .3 barn) . To datefunction –18. For 152Tb (155Tb (149Tb (in vitro and in vivo studies. Similar mass separation techniques have recently been used at the CERN-MEDICIS facility with off-line mass separation capabilities (169Er mass separation proof of concept (up to 17 MBq), obtained from reactor-irradiated enriched 168Er targets, was previously performed using an electromagnetic isotope separation (EMIS) method  method .169Er. The activities produced were sufficient to perform one preliminary in vitro assay. Previously, ionization efficiency values in the order of 20–50% (on mass separators equipped with laser ion sources) were reported for holmium, dysprosium and lutetium  technique to perform precise activity measurements (in vitro experiment was carried out to assess tumor cell viability upon exposure to 169Er-labeled PSMA-617 using cancer cells expressing the prostate-specific membrane antigen (PSMA).This systematic study consisted of several steps, namely, irradiation, mass separation, radiochemistry, quality control, and radiolabeling, to obtain a standard procedure for the production of  therapy . After turements . A preli168Er2O3  was used as target material for the production of 169Er via the 168Er169Er nuclear reaction. The 168Er2O3 samples (7.9–14.2 mg) were sealed in quartz ampoules and irradiated in the V4 irradiation position of the high-flux reactor at Institute Laue–Langevin (ILL), Grenoble, France . After irradiations, the ampoules were transported to the CERN-MEDICIS facility for the offline mass separation of A = 169.Enriched 169Er, supplied as a colloidal suspension of 169Er-citrate from Curium (Swiss distributor: b.e. imaging GmbH), was used as a representative sample for post-irradiation and after chemical separation for activity standardization. The list of all the characterized samples, using different techniques, is given in Carrier-added 169Er. 169Er-containing ampoules were opened and transferred into an ISOLDE standard tantalum target container . The current laser setup consists of two Z-cavity Ti:sapphire lasers, designed by Mainz University   was utilontainer . The coniversity , each puorted in , 48. TheA = 169 beam, containing 169Er, was implanted into a solid catcher (zinc-coated gold foil). Gold foils  were coated with 500 nm Zn  layer using physical vapor deposition (PVD) technique. In total, seven mass-separated samples were shipped to PSI for chemical separation, quality control, and an in vitro proof-of-concept experiment.The mass-separated 169Yb and 169Tm from the desired 169Er. The gold foil obtained from CERN , which is based on tetraoctlyldigycolamide as sorbent. The column was rinsed several times with 6.0 M HNO3 and the Zn concentration in each fraction determined using an Agilent 5110 Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES).The radiochemical separation method was developed for the separation of macro amounts of Zn and as well as isobaric rom CERN  was intr169Yb and 169Er was performed with 0.06–0.08 M α-hydroxyisobutyric acid  using the Sykam cation exchange resin separation system. Subsequently, LN3 resin  was used to remove the complexing agent (α-HIBA), be an additional means of Zn removal and to obtain the final product in chloride form. The use of 2 mL 0.02 M HCl removed the Zn from the system, prior to the elution of 169Er (1 mL 2.0 M HCl).After removal of the macro amounts of Zn; Er, Yb, and Tm were eluted using 0.05 M HCl and loaded onto a column containing Sykam macroporous cation exchange resin , column volume: 0.06 mL], for the complete removal of Zn from the final product. The eluant was, then, heated until dryness and redissolved in 250 μL 0.05 M HCl solution, which was used for the radiolabeling of PSMA-617.As an extra, and final, separation step, Gamma-ray spectra of the gold foils were taken before and after leaching of the Zn layer, using a high-purity germanium (HPGe) detector , in combination with the Inter-Winner software package  .169Er in 0.1 M HCl (stock solution) was divided into two parts, named Er1 and Er2, respectively  (169Er are described in the The 2250 CA) . FurtherAfter chemical separation, the radionuclidic purity of the samples was determined using a HPGe detector, as described in section Activity Standardization.168Er2O3  and four mass-separated 169Er samples (S1-S4) were analyzed using a Thermo Fisher Scientific Sector Field Inductively Coupled Plasma Mass Spectrometer (SF-ICP-MS) Element II®. The measurement solutions were prepared from Suprapur nitric acid  and Milli-Q-water, which were also measured and subtracted as blank. Rhenium standard solution  was added to each measurement solution as an internal standard, to account for plasma instabilities during the measurement. Instrument settings, such as gas flows, were tuned daily.Enriched The measurement was performed in low-resolution mode, scanning the masses 149–170 and for the internal standard 185 and 187. A standard solution containing stable isotopes of rare earth elements, including Er, was measured at the start of the measurement sequence as a performance check.3 (Merck Suprapur), using Sigma-Aldrich TraceCERT®, 1,000 mg/L Er and Zn ICP standards.The chemical purity of the sample was determined using ICP-OES (Agilent ICP-OES 5110). Zn- and Er-containing standard solutions  were prepared in 2% HNO169Er-PSMA-617 obtained was determined using High-Performance Liquid Chromatography (HPLC) with a C-18 reversed-phase column . The mobile phase consisted of MilliQ water containing 0.1% trifluoracetic acid (A) and acetonitrile (B) with a gradient of 95% A and 5% B to 20% A and 80% B over a period of 15 min at a flow rate of 1.0 mL/min. A sample of the radiolabeling mixture was diluted in MilliQ water containing sodium diethylenetriamine-pentaacetic acid  prior to injection into the HPLC instrument.Radiochemical purities of Sample 5 and Sample [6-7 (dissolved together to obtain higher activity)] were determined by radiolabeling, using PSMA-617 as a model compound. The percentage of 169Er has a negligible emission of photon radiation. Therefore, before labeling experiments, the detection of 169Er using a radio-HPLC detector was assessed by comparison to 177Lu. 169Er and 177Lu, respectively. Before injecting the samples, the HPLC column was cleaned by running several blanks using DTPA solution to ensure the detection of 169Er.169Er under standard conditions at a molar activity of 10 MBq/nmol. A stock solution of PSMA-617 (1 mM) was mixed with a solution of 169Er in 0.05 M HCl and the pH adjusted to 4.0 with the addition of sodium acetate solution . The reaction mixture was incubated for 20 min at 95°C. Quality control of 169Er-PSMA-617 was performed using HPLC, as reported above.PSMA-617  was labeled with 177Lu  at a molar activity of 10 MBq/nmol, according to a previously-published procedure  . The cel169Er-PSMA-617 and 177Lu-PSMA-617 (0.01–10 MBq/mL), respectively. The assay was performed according to a previously-published method using a 3--2,5-diphenyltetrazolium bromide (MTT) assay (2) to allow adhesion of the tumor cells. 169Er-PSMA-617 or 177Lu-PSMA-617  were diluted in RPMI medium without supplements to an activity concentration of 0.1–10 MBq/mL. The tumor cells were washed with 200 μL phosphate buffered saline  prior to the addition of the radioligand dilutions (200 μL/well). Control cells were sham-treated with RPMI medium without the addition of radioligands. After a 4 h incubation period , tumor cells were washed with 200 μL PBS and fresh PRMI medium (with supplements) was added to each well. Tumor cell viability was determined after 6 days of incubation at 37°C, 5% CO2. MTT reagent  was added to each well, followed by incubation of the well plates for 4 h. The dark-violet formazan crystals were dissolved with 200 μL dimethyl sulfoxide. The absorbance was measured at 560 nm using a microplate reader . Cell viability was quantified by expressing the absorbance of the test samples as a percentage of the absorbance of untreated tumor cell samples, which was set to 100%.A tumor cell viability assay was performed using PC-3 PIP/flu tumor cells treated with 168Er2O3 were used for 169Er production at the ILL nuclear reactor, with a theoretical yield between 25 and 48 GBq. After the irradiations, the ampoules were transported to the CERN-MEDICIS facility. 169Er (supplied by b.e. Imaging GmbH) as a representative spectrum for post-irradiation. The product had a reported radionuclidic impurity ≤0.38% of the 169Er activity, with 177Lu (0.02%) the main impurity.In total, five ampoules containing 169Er activities were increased up to a factor of four compared to the surface ionization technique (168Er2O3) led to the co-production of 169Yb (t1/2 = 32 d) due to the high thermal neutron capture cross-section of 168Yb (σ: 2400 b) (169Yb (isobar of 169Er) was observed as a radionuclidic impurity  for the two lines at 109.8 and 118.2 keV  as 169Yb  with much lower intensity (8), which made difficult to perform precise 169Er activity measurements of the samples by means of γ-ray spectrometry. Therefore, separation efficiencies were determined based on the theoretical initial and final 169Er activities measured using LSC . Since activity loss during the ampoule opening and transferring were not taken into consideration, the actual separation efficiencies are expected to be higher than the reported values.The mass 169 ion beam was implanted into seven solid catchers (two collections were performed for two ampoules). The mass separation was combined with resonant laser ionization to enhance element selectivity for the erbium ionization, as well as the overall separation efficiency. Thus, echnique . The pre 2400 b) . As a reimpurity . The ave18.2 keV . Moreove169Er activity and the isobar 169Yb, was dissolved from the gold foil. Cation and extraction chromatographic resins were used for the effective separation of 169Er from isobars and macro quantities of zinc to obtain chemically and radionuclidically pure 169Er. DGA extraction resin was used for the first separation step, which required the use of concentrated nitric acid as a loading solution for the separation of macro quantities of Zn from the lanthanides. ICP-OES results showed that traces of zinc (300 ppb) were retained on the resin (169Er and 169Yb using 0.05 M HCl. The separation of lanthanides (Er and Yb) was, subsequently performed using Sykam cation exchange resin. An example of the separation profile is presented in 169Er with high radionuclidic and chemical purity. However, quality control analysis of the final product  showed that a three-step separation process was not sufficient to reach the required high chemical purity. Thus, an additional purification step, using the TK200 resin, was added to separate the remaining Zn from the final product . The uncertainty budget of the measurement is given in 169Er samples were performed using calibrated LSC.In order to perform precise 169Er after chemical separation. Thanks to the effective removal of 169Yb  . The results showed that the tail of  1 and 2 . Sample is (CoA) .169Er-PSMA-617 at a molar activity of 10 MBq/nmol. The radiolabeling of PSMA-617, failed due to the presence of 0.49 μg Zn impurity (determined by ICP-OES) competing with the 169Er for the DOTA chelator of PSMA-617.After the three-step separation process, Sample 5 was used to test the preparation of 177Lu (as a surrogate for 169Er) and DOTANOC in the presence of different masses of non-radioactive Zn  to investigate its therapeutic potential in comparison to the clinically-established 177Lu-PSMA-617. Exposure of PC-3 PIP tumor cells to 169Er-PSMA-617 resulted in reduced tumor cell viability (89 ± 4% and 69 ± 7%) when 5 and 10 MBq/mL, respectively, were applied. At lower concentrations of the applied radioligand (0.1–2.5 MBq/mL) the cell viability was unaffected compared to sham-treated control cells. 177Lu-PSMA-617 had a greater effect on tumor cell viability, resulting in 72 ± 1% and 62 ± 3% viable cells after exposure to 5 and 10 MBq/mL, respectively. Also, tumor cell viability was reduced at lower applied activity concentrations of 1.0 and 2.5 MBq/mL resulting in 80 ± 1% and 79 ± 1% viable tumor cells, respectively , and hence, receptor saturation effects were likely to occur. In order to enable effective cell killing, the radioligand should be prepared at higher molar activity and applied at higher activity concentrations.The radioligand ectively . It is i169Er was thermally ionized (169Er were extracted compared to the previous study (169Er activities were increased up to a factor of four) . Furthermore, the parallel ionization of bulk amounts of 168Er can reduce the ion source performance, impeding the proper separator adjustment. This can be solved with the application of an isotope-selective laser ionization scheme or optimization of the mass separator for high-flux ion-beam operation  , there iperation .169Yb, which was observed by γ-ray spectrometry after the mass separation process. The presence of 169Yb would affect the dosimetric evaluation of the 169Er radiopharmaceutical. Moreover, the chemical purity of the final product would have a direct effect on the radiolabeling of the desired radionuclide to a target molecule (169Er from the isobaric contaminants (169Yb) and soluble residues of the Zn-coated gold foil to make the desired radionuclide available in a purified form suitable for radiolabeling procedures.During the mass separation process, isobaric contaminants can also be extracted due to surface ionization and non-resonant laser ionization. They could also be collected in the Zn-coated gold foils, such as molecule , 57. Thu169Er from 169Yb . It is worth mentioning that the Zn quantity reported in the commercially-available non-carrier-added 177Lu  is ≤0.1 μg/GBq with an activity concentration of 37.5 GBq/mL (80 μL 3 GBq 177Lu can contain 0.3 μg Zn). This quantity of Zn does not affect labeling due to the high activity concentration of the 177Lu solution. However, the activity concentration of the mass-separated 169Er solutions are much lower than the commercially-available non-carrier-added 177Lu solution. Thus, even ppb levels of Zn impurity could affect the labeling efficiency (As mentioned previously, separation of lanthanides is a particularly challenging issue, due to their similar chemical behavior. Previously, Chakravarty et al. used an electrochemical separation method for the complete removal of om 169Yb . The metparation . It was paration –64. In tficiency .169Er with regard to its potential as a therapeutic radionuclide, extensive in vitro and in vivo studies will be necessary. In this regard, the stability of DOTA complexes has to be assessed to confirm stable coordination of 169Er using biomolecules that are currently used with 177Lu. This will, however, only be possible once the radionuclide can be made available in larger quantities and at a quality that allows the preparation of radioligands at high molar activity as it is feasible with other therapeutic radionuclides, including 177Lu and 161Tb.In order to characterize 169Er (E177Lu (Ein vitro assay, however, still fall well short for extensive pre-clinical studies. If the overall efficiencies could be improved from 0.5 to 20% (169Er activities. Moreover, for the present study, irradiations were performed for 7 days. For an industrial production, one could irradiate for longer, minimizing the activity loss during transport and chemical separation, and increasing the yield of 169Er.A typical clinical dose of 5 to 20% , 43–45, 169Er was produced at high specific activities by means of neutron irradiation, followed by mass and chemical separation processes, respectively. By combining surface ionization with resonant laser ionization, 169Er activities were increased up to a factor of four compared to the previously-published study. Further developments of the mass separation process, such as the application of an isotope-selective laser ionization scheme and optimization of the mass separator for high-flux ion-beam operation, are needed to increase the overall separation efficiencies and to provide higher activities of 169Er in future. The availability of the β−-particle-emitting 169Er is particularly important to perform pre-clinical studies, in combination with the pure Auger electron emitter 165Er. This combination will help us explore the additional therapeutic effect of Auger electrons to β−-particles, as in the case of 161Tb, which will certainly be of scientific relevance. Enhanced knowledge of the additive therapeutic effects of Auger electrons will likely be well-received by the nuclear medicine community and ought to pave the way toward more efficient cancer treatment, particularly, for the treatment of single cancer cells and small metastases.In this study, radionuclidically pure The original contributions presented in the study are included in the article/All authors listed have made a substantial contribution to the work, read, and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli showed that the AT-like domain could not restore the growth phenotype of a temperature-sensitive mutant (ts∆fabD) defective in malonyl-CoA:ACP transacylase activity. In vitro assays showed that the AT-like domain possessed thioesterase activity towards a few acyl-CoAs tested where docosahexaenoyl-CoA (DHA-CoA) was the preferred substrate. Expression of this domain in an E. coli mutant (∆fadD) defective in acyl-CoA synthetase activity resulted in the increased accumulation of free fatty acids. Site-directed mutagenesis showed that the substitution of two putative active site residues, serine at 96 (S96) and histidine at 220 (H220), in the AT-like domain significantly reduced its activity towards DHA-CoA and accumulation of free fatty acids in the ∆fadD mutant. These results indicate that the AT-like domain of the PUFA synthase does not function as a malonyl-CoA:ACP transacylase, rather it functions as a thioesterase. It might catalyze the last step of the VLCPUFA biosynthesis by releasing freshly synthesized VLCPUFAs attached to ACP domains of the PUFA synthase in Thraustochytrium.Biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFA) such as docosahexaenoic acid  and docosapentaenoic acid  in protist Thraustochytrium is catalyzed by a polyunsaturated fatty acids (PUFA) synthase comprising three large subunits, each with multiple catalytic domains. This study used complementation test, in vitro assays, and functional expression to characterize an acyltransferase (AT)-like domain in Subunit-B of a PUFA synthase from Thraustochytrium. Complementation test in  Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid DHA, 22:6n-3) and eicosapentaenoic acid EPA, 20:5n-3) are essential components of cell membranes and precursors for eicosanoids and docosanoids involved in mediating various physiological processes in humans and animals . Lack or2:6n-3 ann-3 are eEscherichia coli (E. coli) [Two distinct biosynthetic pathways of VLCPUFAs exist in nature . The aerE. coli) . BiosyntE. coli) .Thraustochytrium sp. 26185 is a unicellular marine protist that produces a high level of DHA. Our recent genome sequencing indicated both aerobic pathway and anaerobic pathway for the biosynthesis of DHA co-existed in Thraustochytrium [E. coli. The results provide evidence that this domain functions as a thioesterase probably involved in releasing freshly synthesized VLCPUFAs as free fatty acids from their thioesters attached to the PUFA synthase in Thraustochytrium.chytrium . Howeverchytrium ,15. The chytrium ,18,19,20E. coli Top10 was purchased from Invitrogen Biotechnology Co. . E. coli fabD mutant (ΔfabD) was obtained from the Coli Genetic Stock Center from the Yale University . E. coli ∆fadD strain was provided by Dr. Pamela Silver, Harvard Medical School  [E. coli SHuffle strain was acquired from New England BioLabs   . E. coliMA, USA) . The pCDresearch . ExpressThraustochytrium sp. ATCC 26185 (GenBank accession no. AOG21005.1) in the National Center for Biotechnology Information website (NCBI) was analyzed by BLASTp (NCBI), MegAlign (DNASTAR), and SWISS-MODEL. The boundaries of the AT-like domain were determined by sequence alignment and homology modeling. Amino acid sequence of the Subunit-B of the PUFA synthase was used as a query to search NCBI protein database by BLASTp with the default algorithm parameters to identify the AT-like domain and its homologous sequences. Three homologous MAT proteins from Streptococcus pneumoniae, Coxiella burnetti, and E. coli were used as templates for homology modelling. Conserved motifs and residues were identified by sequence alignment using the ClustalW method with the slow-accurate option to obtain a precise alignment even if the sequences were highly divergent.Polyunsaturated fatty acid synthase from E. coli MAT was amplified using primers FabD-F and FabD-R  F and AT-like (ORF-B) R, were used to amplify the AT-like domain from ORF-B. Primers MAT (ORF-A) F and MAT (ORF-A) R were used to amplify the MAT domain from ORF-A. d FabD-R . For comΔfabD) defective in fatty acid synthesis was used for the complementation assay [MAT gene was lethal in E. coli [E. coli FabD and pBAD_MAT expressing the MAT domain from Subunit-A of the PUFA synthase, were also included in complementation assays. A single colony of each transformant was first grown in an ampicillin LB liquid medium at 37 °C until an OD600 = 0.4 was reached. After that, 2 μL of each culture were dotted on ampicillin LB plates containing 0.01% (w/v) of l-arabinose [A thermolabile malonyl-CoA:ACP transacylase (MAT) mutant  at the final concentration of 1 mM was used to induce the expression at 16 °C [An at 16 °C . The celw/v) was added to extract any contaminated hydrophilic compounds in the lipid extract. The mixture was centrifuged at 2200 rpm for 10 min, and the aqueous phase was removed as much as possible. The organic solvent phase of extractions was dried under N2 gas [2 gas, total FAMEs were dissolved in 50 μL of hexane and analyzed by gas-chromatography [Ten milliliters of the culture after 24 h of induction were centrifuged at 10,000 rpm for 10 min for collecting cell pellets and supernatant. Total lipids from cell pellets were extracted following the protocol of Folch and colleagues . One milr N2 gas . Free far N2 gas . After ttography .w/w). The organic solvent phase of extractions was dried under N2 gas. Free fatty acids were selectively methylated as described above for cell pellets. After the sample was dried under N2 gas, total FAMEs were dissolved in 50 μL of hexane and analyzed by gas-chromatography. Ten microliters of 0.1 mg/L of heptadecanoic (17:0) were used as an internal standard. All analyses were performed in triplicate.Extracellular free fatty acids extracted from the culture were done by mixing 2 mL of the supernatant with the same volume of hexane and 500 μL of NaCl saturated water , and disrupted through bead beater. The mixture was centrifuged at 15,000 rpm at 4 °C for 15 min. Crude proteins in cell pellets (insoluble fraction) and supernatant (soluble fraction) were separately resuspended in 200 μL of SDS-PAGE loading buffer  for electrophoresis [E. coli SHuffle transformed with pET28a empty vector.phoresis . The negThioesterases catalyze the release of acyl moieties from the phosphopantetheine of ACP or CoA . As acylE. coli SHuffle and E. coli ∆fadD strains for enzymatic and free fatty acid analysis.Overlapping PCR was employed for the site-directed mutagenesis of two residues, S96 and H220, separately, in the AT-like domain. The mutagenesis involved two sequential PCR reactions using two flanking primers (AT-like F and R) and two internal primers  to introduce the mutation . The intp < 0.05) with multiple comparisons. Additionally, an unpaired t-test at p < 0.05 was also used to compare activities between SHuffle control and SHuffle AT-like expression on each substrate.The statistical analysis of the data from three independent biological replicates were performed using a one-way ANOVA . However, it shares low amino acid identity to discrete MAT proteins from E. coli [Coxiella burnetti [Streptococcus pneumoniae [Amycolaptosis mediterranei [Streptomyces coelicolor [PUFA synthase for the biosynthesis of DHA in  domains . A MAT d domains . All the domains . Two higrium sp.  and Aura E. coli , Coxiellburnetti , and Streumoniae   elicolor  (16.4%).E. coli fabD mutant defective in malonyl-CoA:ACP transacylase (MAT) activity. As MAT is essential for fatty acid synthesis; only a temperature-sensitive mutant of this gene is available in E. coli for the complementation test. The wild-type strain (WT) with an empty vector could grow at both permissive (37 °C) and non-permissive temperatures (42 °C), while the mutant with the AT-like domain from Subunit-B (Mutant+AT-like), similar to the mutant with the empty vector (Mutant), could grow at 37 °C, but not at 42 °C. On the other hand, the mutant with either discrete E. coli MAT gene FabD (Mutant+FabD) or the authentic MAT domain from Subunit-A (Mutant+MAT) could effectively grow at both 37 °C and 42 °C , but neither is essential. No changes on growth phenotype and fatty acid profile could be detected in a double mutant (∆tesAtesB) [As the AT-like domain did not function as malonyl-CoA:ACP transacylase, next we attempted the complementation test of this domain in esAtesB) . To inteE. coli mutant (fadD) defective in acyl-CoA synthetase activity was exploited to express the domain , octadecenoyl-CoA (18:1-CoA), octadecadienoyl-CoA (18:2-CoA), and docosahexaenoyl-CoA (DHA-CoA). The highest activity was found towards DHA-CoA, followed by 18:2-CoA, 18:0-CoA and 18:1-CoA after normalization against the control  defective in acyl-CoA synthetase activity confirms that the mutated AT-like domains are not functional, as the amounts of free fatty acids in the mutant strain expressing the two mutated domains are even lower than those in the strain with the empty vector. The lower production of free fatty acids in the transformants with the mutated AT-like domain could be due to the dominant negative mutation effect [E. coli thioesterases and non-functional heterologous mutated AT-like domains could compete for the same substrate, leading to the reduced production of free fatty acids.Sequence analysis using a combination of bioinformatic tools indicates the AT-like domain resides between 1048 and 1390 amino acids in the Subunit-B of the PUFA synthase. A GXSXG motif and a histidine residue are conserved among the AT-like domain and related sequences, such as MAT and thioesterase proteins. The conserved serine at position 96 and the histidine at position 220 might be part of a catalytic triad in the active site for catalysis , as the n effect , as funcE. coli temperature sensitive MAT mutant (ts∆fabD). This mutant has been previously used to examine the function of predicted MAT proteins by complementation tests [E. coli double mutant (ΔtesAtesB) was employed to express the domain, as it has the second highest sequence similarity to thioesterases. However, expression in the mutant did not produce any detectable changes on growth phenotype and lipid/fatty acid profile, similar to the previous result that no difference in growth and fatty acid profile could be observed between the mutant and wild type strain [E. coli mutant (ΔfadD) where acyl-CoA synthetase gene was deleted. This mutant has been widely used for enhancing the production of free fatty acids in bacteria [E. coli produces fatty acids principally for the biosynthesis of membrane lipids and normally does not accumulate a high level of free fatty acids [To functionally analyze the AT-like domain, it was first expressed as a standalone protein in an on tests . The rese strain . Subsequbacteria ,48,49,50E. coli fabD mutant was first used to show that the AT-like domain of a PUFA synthase from Thraustochytrium does not function as a MAT. The increased production of free fatty acids in an E. coli acyl-CoA synthetase mutant expressing the domain then indicated thioesterase activity by releasing free fatty acids from their thioesters. In vitro enzymatic assays using the domain protein expressed in the E. coli SHuffle strain as an enzyme source ultimately confirmed that this domain could hydrolyze fatty acyl-CoA substrates with the highest activity towards a thioester of DHA. To prepare the domain protein for in vitro activity assays, we first attempted the expression in a high expression system BL21 (DE3) or Rosetta (DE3). However, all the expressed proteins in these strains appeared in the insoluble fraction (E. coli strain Shuffle, which has enhanced capacity for appropriate folding. With this strain, we were able to show some activity by in vitro assays using the crude protein. Although the activity is low, the higher activity value of the transformant relative to the empty vector control would indicate the authentic catalytic mode. Collectively, these results from in vitro assays, complementation test, mutagenesis analysis, and production of free fatty acids in an E. coli ΔfadD mutant support the notion that the AT-like domain of the PUFA synthase functions as a thioesterase. This activity is probably involved in catalyzing the last step of the VLCPUFA biosynthesis by releasing freshly synthesized DHA attached to ACP domains of the PUFA synthase into a pool of free fatty acids for subsequent metabolisms in Thraustochytrium.In marine Thraustochytrids, VLCPUFAs are solely synthesized by an anaerobic pathway catalyzed by a PUFA synthase ,51. Aftefraction . This di


Preeclampsia is a severe placenta-related pregnancy disorder that is generally divided into two subtypes named early-onset preeclampsia (onset <34 weeks of gestation), and late-onset preeclampsia (onset ≥34 weeks of gestation), with distinct pathophysiological origins. Both forms of preeclampsia have been associated with maternal systemic inflammation. However, alterations in the placental immune system have been less well characterized. Here, we studied immunological alterations in early- and late-onset preeclampsia placentas using a targeted expression profile approach. RNA was extracted from snap-frozen placenta samples . The expression of 730 immune-related genes from the Pan Cancer Immune Profiling Panel was measured, and the data were analyzed in the advanced analysis module of nSolver software (NanoString Technology). The results showed that early-onset preeclampsia placentas displayed reduced expression of complement, and toll-like receptor (TLR) associated genes, specifically TLR1 and TLR4. Mast cells and M2 macrophages were also decreased in early-onset preeclampsia compared to healthy placentas. The findings were confirmed by an immunohistochemistry approach using 20 healthy, 19 early-onset preeclampsia, and 10 late-onset preeclampsia placentas. We conclude that the placental innate immune system is altered in early-onset preeclampsia compared to uncomplicated pregnancies. The absence of these alterations in late-onset preeclampsia placentas indicates dissimilar immunological profiles. The study revealed distinct pathophysiological processes in early-onset and late-onset preeclampsia placentas and imply that a tailored treatment to each subtype is desirable. Problems of placental development and adaptation can result in impaired fetal growth and preeclampsia affecting 2-8% of all pregnancies . The serAlthough the etiology is incompletely understood, preeclampsia is generally considered a condition of placental insufficiency, accompanied by an exaggeration of the normal pregnancy-induced maternal systemic inflammatory response . The immwww.nanostring.com). A high sensitivity of this technique allows the identification of non-abundant immune cells, even in diseases with an immune-suppressive microenvironment like pancreatic cancers , thought to originate from deficient placentation, and late-onset preeclampsia (onset ≥34 weeks of gestation), believed to be predominantly driven by maternal cardiovascular risk factors and placental insufficiency . Distinc22.1All patients who were admitted to the Erasmus MC in Rotterdam gave written approval to participate in this study, prior to giving birth. The participants were either patients with singleton term pregnancies undergoing elective caesarean section or patients with preeclampsia, diagnosed based on the new ISSHP 2018 criteria, i.e., new-onset hypertension after 20 weeks of gestation (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg), accompanied by maternal organ dysfunction (e.g. proteinuria as defined by urinary protein/creatinine ratio >30 mg/mmol) and/or uteroplacental dysfunction defined as fetal growth restriction . Early-o2.2Within 20 minutes after delivery of the placenta, biopsies were taken from areas without obvious infarcts/necrosis, snap-frozen and stored at -80°C. In a limited number of samples from the same population, one additional full-thickness slice (1 cm in thickness) was collected per placenta and fixed in formalin, then embedded in paraffin (FFPE). Since FFPE samples were not available from all patients included in the gene expression profiling measurements, the number of FFPE samples was expanded as described in the previous section to confirm the results by immunohistochemistry.2.3For RNA extraction, frozen biopsies were homogenized in RLT lysis buffer  with β-mercaptoethanol. After proteinase K  treatment for ten minutes at 55°C, RNA was extracted with the Qiagen RNA easy mini kit  according to the manufacturer’s instructions, eluted in RNA free water, and stored at -80°C. RNA Quality Control (QC) was measured using the 2100 Bioanalyzer . NanoString Technology is based on detecting the genes of interest using 100 base pairs, but a cut-off value of 300 base pairs is necessary to increase the accuracy of measuring all the genes. Therefore, the total RNA concentration was corrected to include fragments seized between 300 and 4000 nucleotides. A total of 200 ng RNA was hybridized to the PanCancer Immune Profiling Panel for 17 hours at 67°C, following the manufacturing procedure . The nCounter FLEX platform was used and genes were counted by scanning 490 Fields-of-view (FOV).2.3.1Data analysis was performed using the advanced analysis module (version 2.0) of nSolver™ software . The quality control of the measurements was done according to the general workflow used in the NanoString nCounter software analysis . Based o2 transformed and the differentially expressed genes were identified using simplified negative binomial models, mixture negative binomial models, or log-linear models based on the convergence of each gene. The adjusted P-value was calculated using the Benjamini-Hoghberg method. Genes were considered differentially expressed when the adjusted P-value < 0.05 and the linear fold of change (FOC) > 1.5 in any direction.To identify differentially expressed genes in preeclampsia samples, healthy placenta samples were used as controls. Hence, data of the early-onset preeclampsia samples and that of the late-onset preeclampsia samples were compared to healthy control samples. In addition, to investigate the differences between the two groups of preeclampsia, data of late-onset preeclampsia were compared to that of early-onset preeclampsia. The normalized data were log2.3.2Specific and unique cell type marker genes were used to define multiple immune cell subtypes –27. By u2.3.3Pathway alterations were analyzed by clustering genes into pathways using the default settings of the PanCancer Immune Profiling advanced analysis module in nSolver. The pathway scores were calculated for each sample as the square root of the average squared t-statistic of all genes in the corresponding pathway.P-value < 0.05, comparing early-onset preeclampsia and late-onset preeclampsia to healthy placentas. Genes that were related to either TLR signaling, the complement pathway or chemotaxis-associated cytokines as identified in DAVID were used to perform unsupervised hierarchical heatmap clustering using Ward’s method and visualized using the ComplexHeatmap package in R  v6.8 –31 was uage in R .2.42.4.1Sections of 4µm of FFPE samples were used for immunohistochemistry analysis. To visualize M1 and M2 macrophage subsets in placental tissue, the chromogenic duplex staining was done with CD68 and CD163 by an automated staining procedure using the Ventana Benchmark Discovery . In brief, following deparaffinization and heat-induced antigen retrieval with CC1  for 40 minutes at 95°C, the tissue samples were firstly incubated with CD68 for 60 minutes at 37˚C, followed by detection with anti-mouse HQ  for 16 minutes and subsequently anti-HQ HRP (#760-4820), and visualized with Discovery Purple  for 32 minutes. An antibody denature step was performed using CC2  for 20 minutes at 100˚C. Secondly, CD163 was incubated for 32 minutes at 37˚C, followed by detection with anti-mouse HQ and subsequently anti-HQ HRP, and visualized with Discovery Teal  for 32 minutes. Finally, all slides were counterstained with hematoxylin II (#760-2208) and bluing reagents (#760-2037) for 4 minutes. Antibody information and clonality can be found in 2.4.2To visualize mast cells in placental tissue, sequential 4 µm thick (FFPE) sections were stained by conventional immunohistochemistry for tryptase, using the optiview universal DAB detection kit . In brief, following deparaffinization and heat-induced antigen retrieval with CC1  for 64 minutes, the tissue samples were incubated with the antibody of interest . The number of positive cells with visible nuclei was counted by hand. In the central areas of the placenta, only cells inside the villi were counted, excluding cells in the intervillous space. To normalize for variation in decidual thickness, the decidual area was drawn by hand, measured, and cell counts were reported as cells/mm2. For the combined CD68/CD163 staining, cells were counted as either double-positive cells or CD68 single-positive cells. These cells were predefined as M2, and M1 macrophages, respectively. All analyses were performed blinded by one researcher. Power analysis for immunohistochemistry revealed a requirement of n=20 per group to obtain statistical significance at α=0.05 and β=0.2. However, there was not enough late-onset preeclampsia material available, and one early-onset preeclampsia placenta had too many infarcts and was therefore excluded.For analysis, the immunohistochemistry slides were scanned on a NanoZoomer 2.0-HT (Hamamatsu Photonics K.K.). In NDPview (version 2.5.19 Hamamatsu Photonics K.K.), 1 mm2.5P-value < 0.05.Unless stated otherwise, analyses were conducted and images were produced in Graphpad Prism 8 or R 4.1 using the ggplot2 package , 34. The33.1Out of the 36 mRNA samples which were measured by the gene expression array, four samples had to be excluded from further analysis. One sample was retrospectively excluded due to misclassification as late-onset preeclampsia while the patient suffered from pregnancy-induced hypertension. Three healthy control samples were excluded because these did not meet the quality control criteria after gene expression measurement. The binding density in these samples, reflecting the total percentage of detected genes, was <0.2 compared to >0.7 in the 32 included samples, this was also reflected by significantly lower counts of positive controls and housekeeping genes Table 1.3.2Analysis of 730 individual genes revealed 33 downregulated, and 15 upregulated genes in early-onset preeclampsia compared to healthy controls (3.3P=0.005), mainly due to reduced expression of TLR4 and TLR1  and mast cells  were decreased in early-onset preeclampsia samples, while M1 macrophages (CD86+MSR1+) were not altered based on gene expression (P=0.015), it did not differ in either condition when compared to healthy placentas. All other investigated cell types did not statistically significantly differ between our groups.Using the gene expression analysis, we identified multiple immune cell subtypes in the placenta  syndrome in both subtypes. Nevertheless, the results of this study indicate that a maternal systemic inflammatory state does not necessarily translate to an inflammatory placenta.de novo NAD+ synthesis, and is involved in vascular development and functioning, anti- and pro-oxidative processes, and immune regulation. However, the function of the kynurenine pathway is mainly determined by tryptophan transporters  repository, accession number GSE190639, The study was reviewed by the Medische Ethische Toetsings Commisie Erasmus MC and exempted from medical approval according to the Dutch Medical Research with Human Subjects Law (MEC-2016-418 and MEC-2017-418). The participants provided their written informed consent to participate in this study.The study was designed by MB, IR, and DM. Data collection was performed by MB, EH, TB, and JD, whilst data analysis was done by MB and DM. MB, EH, RV, BR, and CE contributed to the data interpretation. MB wrote the manuscript and designed the figures. All authors contributed to the article and approved the submitted version.www.supportcasper.org).This work was partially supported by the Support Casper Foundation (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Noncoding RNA (ncRNA) is involved in the occurrence, development, metastasis, and drug resistance of tumors and involves a variety of biological functions. In addition, miRNA can regulate proliferation and migration and even regulate epigenetics to promote the development of multiple myeloma (MM). However, the mechanism of ncRNA involved in MM is still unclear, and there are many unknown ncRNAs to be explored. This research is aimed at discovering the unknown lncRNA in MM through high-throughput sequencing and to study the mechanism and role of competitive endogenous RNA (ceRNA) involved in the pathogenesis of MM for the development of novel molecular markers and potential new targeted drugs. We screened out 262 new lncRNAs with statistical differences by RNA sequencing and selected the lncRNA MSTRG.29039.1 according to the expression and function of lncRNAs and their target genes in MM. We verified that MSTRG.29039.1 and its target gene OSMR were highly expressed in MM. After knockdown of MSTRG.29039.1 in MM cell lines, the expression of OSMR was decreased, and the expression of hsa-miR-12119 was upregulated which can also promote cell apoptosis and inhibit proliferation. Then, we knocked down hsa-miR-12119 and MSTRG.29039.1, we found that apoptosis of MM cells was reduced, and cell proliferation was increased compared with just knocking down hsa-miR-12119. We further verified the direct binding relationship between MSTRG.29039.1 and OSMR by the dual-luciferase reporter assay system. Thus, MSTRG.29039.1 can competitively bind with miRNA to counteract the inhibitory effect of miRNA on OSMR, which regulates cell proliferation and apoptosis through the JAK2/STAT3 pathway. In a conclusion, lncRNA MSTRG.29039.1 could promote proliferation by sponging hsa-miR-12119 via the JAK2/STAT3 pathway in multiple myeloma. This may be a molecular marker and a potential therapeutic target for MM. Multiple myeloma (MM) is a plasma cell disease characterized by biological heterogeneity whose clinical features are clonal hyperplasia of bone marrow plasma cells and accompanied by excessive production of monoclonal immunoglobulins, leading to a series of damages such as abnormal kidney function, hypercalcemia, anemia, and bone disease . With thWith the rapid development of high-throughput technologies, we have gradually discovered that noncoding RNA (ncRNA) plays an important role in the pathogenesis of myeloma. Long noncoding RNA (lncRNA) is RNA that is more than 200 nucleotides in length and has little or no ability to synthesize proteins . A largeμL PBS twice, added the cell suspension to collect the negative cells. Wash 1-2 times with 500 μL PBS buffer as well as removing the MS column from the magnetic field before adding 500 μL PBS  buffer and quickly beating it into the collection tube. At last, count the number of cells and add 700 μL of TRIzol  per 107 cells, storing the specimen at -80°C for later RNA extraction. We selected the CD138 cells of 2 healthy volunteers and 8 NDMM patients to operate high-throughput sequencing. All patients and volunteers included in the study signed an informed consent form and recorded their clinical characteristics. The study was approved by the local ethics committee. The collection of the above samples was reviewed and approved by the Ethics Committee of Tianjin Medical University General Hospital, with informed consent from both patients and healthy controls and signed informed consent voluntarily.Bone marrow was collected from 30 healthy volunteers as well as 37 newly diagnosed MM (NDMM) patients diagnosed in the General Hospital of Tianjin Medical University. There were 37 patients with NDMM in the group, including 22 males and 15 females, with a median age of 63 years (39-96 years). Among them, 22 patients with NDMM were less than 65 years old (59.45%), and 15 patients were over 65 years old (40.55%). The normal control group consisted of 30 healthy controls who matched the age and sex of the NDMM group, with a male-to-female ratio of 17/13 and a median age of 54 years (30-84 years) . And theμL). The real-time fluorescent quantitative PCR system was 25 μL, and the reaction was carried out under the following conditions: 95°C, 3 min; 95°C, 5 s, annealing temperature. Primer sequences were shown in We separated RNA by TRIzol, chloroform, isoamyl alcohol, and ice ethanol  and used fast reverse transcription kit  to perform reverse transcription to obtain complementary DNA. Then, we prepared a reverse transcription reaction system  differential mRNA data, while for the prediction of transregulation, firstly, extracted the sequence of differentially expressed lncRNA and mRNA, then used blast software for initial screening (e < 1E − 5), and at last used RNAplex software to screen again to identify possible target genes of lncRNA.The experimental sequencing process was as follows: after extracting the total RNA of the sample, the rRNA was removed using a ribosomal kit, and the RNA was fragmented (the average fragment length is about 200 nt). Reverse transcription to synthesize single-stranded cDNA before synthesizing and purifying double-stranded cDNA and then repaired the ends and added adaptor primers to amplify and purify by PCR. Finally, we performed quality inspection on the library and sequence on the computer. For the detected mRNA, we further calculated the gene expression and analyzed the differentially expressed genes between samples and perform Gene Ontology (GO) analysis and KEGG biological pathway enrichment analysis on the differentially expressed genes. For the detected lncRNA, analyzed the differential expression of lncRNA between samples and gave specific annotations to perform the corresponding identification of ORF protein domain, coding potential, secondary structure prediction, and family analysis. The potential The three software miRanda, PITA, and RNAhybrid  were used to predict candidate miRNAs that interacted with lncRNA and their targeted regulated genes. The prediction of the analysis of interaction with lncRNA mainly considered the following 4 points: [1] the degree of perfect complementary pairing of the miRNA seed region, [2] MRE (miRNA recognition elements) sequence conservation, [3] the binding free energy of miRNA-lncRNA duplex, and [4] the sequence characteristics of the target molecule are determined. We predicted the recognition region of lncRNA and miRNA by miRanda, PITA, and RNAhybrid to identify the lncRNA that interacted with miRNA.2.Human MM cell lines   were cultured in RPMI-1640 medium  and added with 10% fetal bovine serum  and 1% chain penicillin (Gibco), placed in a humid 37°C cell incubator with 5% CO5 cells per well. Use Lipofectamine 3000  reagent to perform cell transient transfection according to the manufacturer's instructions. For si-RNA, miRNA-inhibitor, fluorescence, and negative control were purchased from GenePharma  at 450 nm wavelength was measured using the automatic microplate reader, and the cell proliferation ability was expressed as A value. Each group had 5 replicate wells, and each experiment was repeated 3 times. Cell survival rate = / × 100%.Use Cell Counting Kit-8  for measurement. Inoculate 10Washing the MM cells collected after transfection with 1 : 9 configured phosphate buffer solution , collect the cells after centrifugation at 1500 rpm for 5 minutes. Follow the instructions of the PE Annexin V cell apoptosis detection kit . Finally, the apoptotic cells were distinguished by flow cytometry, and the apoptosis rate was calculated. Perform three replicate measurements.The MSTRG.29039.1 fragment with the predicted binding site of hsa-miR-12119 was synthesized and cloned into the luciferase reporter gene to form the reporter vector MSTRG.29039.1-wild-type (MSTRG-WT). MSTRG.29039.1-hsa-miR-12119 binding site was mutated as instructed and named MSTRG.29039.1 mutant (MSTRG-MUT). OSMR-wild-type (OSMR-WT) and OSMR-mutated type (OSMR-MUT) were also obtained in accordance with the above method. MSTRG-WT/OSMR-WT/MSTRG-MUT/OSMR-MUT/miRNA-mimics/miRNA-NC  were transfected into HEK-293T cells according to the above transfection method. 48 hours after transfection, the dual life luciferase assay system  was applied according to the prescribed procedure. Perform three replicate measurements.After 72 h of transfection, the cells were collected into 1.5MLEP tubes. The cells were lysed with protein lysates consisting of RIPA: PMSF = 100 : 1. The protein concentration was determined using the BCA protein assay kit. After adding the protein sample buffer, the cells were boiled in a 100°C- water bath for 10 min to denaturate the proteins.Start electrophoresis after adding protein and maker to the sample tank. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane . PVDF membrane was cut according to the molecular weight of the required proteins. And then the PVDF membrane was sealed in 5% skimmed milk powder for 1 h and washed with TBST for 3 times. The antibody   was incubated and placed in a shaker for 4°C overnight, and the anti-rabbit IgG sheep antibody  incubated for exposure the next day. The chromogenic condition of the protein was observed and photographed.U test was used to compare the differences between the two groups. The Kruskal-Wallis H test was used for comparison between multiple groups. P < .05 was considered statistically significant.Use SPSS software 22.0 and GraphPad Prism5.0 to complete the data analysis. All data were expressed as the mean ± SD obtained in three repeated determinations. The Mann-Whiney P < 0.05 and fold change > 2 to elect the differentially expressed genes and detected 285 differential genes that were statistically different, 92 were upregulated, and 193 downregulated  to obtain signal transduction and disease pathways. This analysis was mainly concentrated in metabolism, human diseases, environmental information processing, and cellular processes, such as fatty acid biosynthesis, hepatitis C, various cancer pathways, FoxO signaling pathway, endocytosis,and lysosomes. Gene Ontology (GO) analysis can annotate the function of each gene and calculate the most significant function in a specific series of genes through statistical analysis such as hypergeometric distribution. This analysis mainly showed significant differences in cellular components, among which the cell part and intracellular part were the most significant.From the upregulated 203 lncRNAs, we selected 63 lncRNAs that were highly expressed in 8 myeloma patients and submitted them for testing to predict their target genes and the GO function of the target genes and KEGG biological pathway enrichment analysis Figures . BiologiP < .05), and the expression level in MM cell lines  was also significantly higher than that in healthy controls , while statistically significant with ISS staging (P < .05) (β2-microglobulin (β2-MG) (P < .01), plasma cell of biopsy (P < .01), and lactate dehydrogenase (LDH)(P < .05)    and posi Figures . Based oP < .05) .To explore the role of MSTRG.29039.1 in MM, we used si-MSTRG to knock down MSTRG.29039.1 in U266 and AMO-1 cell lines. The transfection efficiency was first confirmed by real-time PCR . PCR andWe found that in a variety of tumors such as pancreatic ductal adenocarcinoma, glioblastoma, and colorectal cancer, OSMR is highly expressed, and OSMR can regulate cell proliferation and tumor growth –21. To sP < .01, P < .05; U266: P < .05, As increasing lncRNAs are discovered, the complexity and diversity of potential ceRNA interactions have increased exponentially. Studies have found that some lncRNAs may be naturally effective spongy miRNAs in some cases . We use To further verify whether MSTRG.29039.1 sponges hsa-miR-12119 to counteract the inhibitory effect on OSMR, we further carried out a rescue experiment. Firstly, after knocking down MSTRG.29039.1 in the U266 and AMO-1 cell lines, we detected that the expression of hsa-miR-12119 increased at the RNA level, while the expression of OSMR decreased. After knocking down hsa-miR-12119 at the same time, the expression of hsa-miR-12119  decreaseWe further studied whether knocking down hsa-miR-12119 can rescue the apoptosis and proliferation pathways regulated by OSMR. After knocking down MSTRG.29039.1, the apoptosis rate of the si-MSTRG group as described above was higher than that of the NC group, and the cell survival rate was reduced in the U266 and AMO-1 cell lines. In the U266 cell line, when we knocked down hsa-miR-12119 and MSTRG.29039.1 at the same time, we found that the apoptosis rate dropped from 34.51% ± 2.95 to 22.96% ± 2.01 at 24 h; at 48 h and 72 h, the apoptosis rate of the si-MSTRG group vs. si-MSTRG+miRNA-inhibitor group was 44.14% ± 3.02 vs. 32.76% ± 1.00 and 53.56% ± 2.32 vs. 39.69% ± 1.50 . CompareThrough the above experiments, we have gradually proved that MSTRG.29039.1 competes with OSMR to bind hsa-miR-12119, thereby reducing the inhibitory effect of hsa-miR-12119 on OSMR. OSMR can regulate cell apoptosis and proliferation, thereby affecting tumor cells and promoting tumor development. However, we still do not know how OSMR affects cell proliferation and apoptosis in MM.The activation of JAK kinase can lead to phosphorylation and dimerization of tyrosine or serine residues in the c-terminal domain of the STAT3 protein. The activated STAT3 dimer enters the nucleus and initiates the transcription of target genes , 24. AndMultiple myeloma is the second most common malignant tumor in the blood disease system. The standardized prevalence and incidence rates in our country are 5.68 (5.64-5.72) and 1.15 (1.11-1.19), respectively, while those in Western Europe and North America are even higher . With thδ, and the main JAK/STAT pathways [OSM is a member of the interleukin 6 (IL-6) receptor family, which is produced by monocytes/macrophages, dendritic cells, and T lymphocytes . Oncostapathways . In gastpathways . The ovepathways . We veriThe abnormal expression of miRNA is also closely related to the disease , 37. Of In summary, our results indicate that MSTRG.29039.1 is highly expressed in MM patients and can potentially competitively bind to hsa-miR-12119, leading to the overexpression of OSMR, reducing apoptosis, and promoting proliferation. We discovered the novel lncRNA MSTRG.29039.1 and found that it plays an important role in the occurrence and development of MM, and it is expected to become a molecular marker and potential therapeutic target for MM.


Training a non-dominant hand is important for rehabilitating people who are required to change handedness. However, improving the dexterity in using chopsticks with a non-dominant hand through training remains unclear. This study is aimed to measure whether chopstick training improves non-dominant hand chopstick operation skills and leads to acquisition of skill levels similar to those of the dominant hand.This single-blinded randomized controlled trial enrolled 34 healthy young right-handed subjects who scored >70 points on the Edinburgh Handedness Questionnaire Inventory. They were randomly allocated to training or control groups. The training group participated in a 6-week chopstick training program with the non-dominant left hand, while the control group did not. Asymmetry of chopstick operation skill, perceived psychological stress, and oxygen-hemoglobin concentration as a brain activity measure in each hemisphere were measured before and after training.F ≥ 5.54, p ≤ 0.03, partial η2 ≥ 0.156). Only perceived psychological stress had a significantly higher asymmetry during the post-training assessment .Participants in the training group had significantly lower asymmetry than those in the control group during the post-training assessment (Six weeks of chopstick training improved non-dominant chopstick operation skills, and a performance level similar to that of the dominant hand was acquired. Approximately 70–80% of stroke patients have upper extremity paralysis, which compromises the motor skills necessary for activities of daily living ,2. Only The dominant hand has better performance speed of manual dexterity ,4,5, accSeveral studies have shown the effectiveness of tool-use training of the non-dominant hand ,10,11. PAn important method for supporting the assessment of skill acquisition is to evaluate perceived psychological stress which, if experienced during task performance, can impair efforts to be physically active and motivated ,14. StreWe have previously reported on training-derived changes in brain activity using the non-dominant left hand . The stu22.1This study was a single-blinded randomized controlled trial with stratification. Participants who met the inclusion criteria were randomly allocated to either the training or the control group by a researcher who was neither an evaluator nor a trainer. The training group received 6 weeks of training with the non-dominant hand with chopsticks for 30 min every day, 5 days per week. The control group did not receive any training.The study was registered at clinicaltrials.gov, trial registration (Registered number: UMIN000044007).2.2The training group participants used their left non-dominant hand to repeatedly transfer various objects  of different sizes, weights, shapes, and materials from one dish to another with 21-cm long wooden chopsticks. All participants in the training group practiced operating the chopsticks continuously at our laboratory for 30 min a day under the surveillance of the trainers.The experimental procedure followed in this study is illustrated in The smoothness of upper extremity movement, perceived psychological stress, and brain activity were evaluated during the marble transferring task. The details of the experimental procedure, chopstick operation task, and functional near-infrared spectroscopy (fNIRS) are described in our previous study .2.3d = 1.09) was calculated based on a previous study that performed chopstick operation training with the non-dominant hand  = −0.47, p = 0.64; sex: χ2 = 0.00, p = 1.00; Handedness Questionnaire Inventory score: t[30] = 0.001, p = 0.99).There were no significant differences in age, sex, and Handedness Questionnaire Inventory score between the training and control groups  and a significant time × group interaction  (post-hoc t-test showed that the asymmetry in the task completion time of the training group (−4.87 ± 25.12) was significantly lower than that of the control group (29.83 ± 35.48) during the post-training assessment , indicating an increased speed of execution of object transfer with the non-dominant hand chopstick operation that approached the speed of the dominant hand. The planned contrasts revealed no significant differences in the training group post-training assessment   as the within-subject factor and group (training group vs control group) as the between-subjects factor, revealed a significant main effect of group (= 0.156) . A post- = 0.45) .3.3F = 17.76, p < 0.001, partial η2 = 0.372) and a significant time × group interaction  (post-hoc t-test showed that the asymmetry of RMSJ in the training group (−1.32 ± 3.76) was significantly lower than that in the control group (2.70 ± 6.02) during the post-training assessment , indicating an improved smoothness in movement of the non-dominant hand for chopstick operation that was close to the smoothness in movement of the dominant hand. The planned contrasts revealed no significant difference in the training group during the post-training assessment   as the within-subjects factor and with a group (training group vs control group) as the between-subjects factor, we found a significant main effect of time (= 0.188) . A post- = 0.18) .F = 8.17, p < 0.01, partial η2 = 0.214) and a significant time × group interaction . A post-hoc t-test showed that the asymmetry of the RMSJ in the training group (−0.95 ± 2.37) was significantly lower than that in the control group (1.93 ± 2.89) during post-training assessment , indicating an increased smoothness in movement with the non-dominant hand chopstick operation, bringing it close to that of the dominant hand. The planned contrasts revealed no significant difference in the training group during post-training assessment   .F = 10.87, p < 0.01, partial η2 = 0.266) and a significant time × group interaction . A post-hoc t-test showed that the asymmetry of RMSJ in the training group (−0.95 ± 3.56) was significantly lower than that in the control group (3.28 ± 5.77) during post-training assessment , indicating an increased smoothness in movement with the non-dominant hand chopstick operation, bringing it close to that of the dominant hand. The planned contrasts revealed no significant difference in the training group during post-training assessment   .3.4F = 65.50, p < 0.001, partial η2 = 0.686) and a significant group × time interaction  (post-hoc t-test showed that the asymmetry of the RMSJ in the training group (0.98 ± 1.04) was significantly lower than that in the control group (2.71 ± 2.32) during the post-training assessment , indicating decreased perceived psychological stress with the non-dominant hand chopstick operation that was close to the perceived psychological stress of the dominant hand. The planned contrasts revealed a significantly higher asymmetry than 0 in the training group during post-training assessment   as the within-subject factor and group (training group vs control group) as the between-subjects factor, revealed a main effect of time (= 0.629) . A post- < 0.01) .3.5F = 4.69, p = 0.04, partial η2 = 0.135), indicating that the brain activity during left-hand chopstick performance was significantly higher than that during right-hand chopstick performance. However, there was no other significant main effects or interactions  (In a 2 × 2 × 2 mixed-design ANOVA of the Oxy-Hb concentration change in the left and right hemispheres with time (pre-training vs post-training) and chopstick use (right hand vs left hand) as the within-subject factors and with a group (training group vs control group) as the between-subjects factor, we found only a significant main effect of chopstick use in the right hemisphere (l cases) .3.6r = 0.54, uncorrected p = 0.03). The change in the asymmetry of RMSJ in elbow flexion-extension was positively correlated with that of shoulder flexion-extension  and shoulder abduction-adduction  (We found that the change in the asymmetry of VAS was positively correlated with that of the Oxy-Hb concentration in the left hemisphere ( < 0.01) .4A 6-week chopstick training program improved chopstick operation skills, decreased perceived psychological stress, and changed the brain activity. Planned contrasts had no significant differences from the value of zero for mean asymmetry in the marble transferring task and the smoothness of targeted joint movements during the post-training assessment. These results suggest that chopstick operation training of the non-dominant hand improved speed and smoothness of joint movement and nearly reached the performance level of the dominant hand. Moreover, there was no individual joint movement-specific delayed skill acquisition or inadequate patterns. These findings complement other studies with similar findings ,9,10. PhIn the untrained dominant hand, speed, smoothness, and perceived psychological stress did not significantly improve between pre- and post-training assessments; thus, there was no inter-limb transfer effect between the non-dominant hand chopstick training in contrast to previous studies ,9,10. ThWe identified the training-derived hemispheric specialization change based on the ROI analysis. We found no significant interaction between Oxy-Hb concentrations in each hemisphere. There was a significantly higher Oxy-Hb concentration in the non-dominant hand than in the dominant hand in the right hemisphere. Previous studies reported that increased ipsilateral hemispheric activity during motor tasks was observed only in the non-dominant hand, while only greater contralateral hemispheric activity was observed in the dominant hand ,37,38,39The changes in asymmetry of chopstick operation skill, psychological stress, and Oxy-Hb concentration revealed positive correlations between changes in the asymmetry of VAS and changes in Oxy-Hb concentration in the left hemisphere and between changes in the asymmetry of RMSJ in elbow flexion-extension and those of shoulder flexion-extension and shoulder abduction-adduction. The close relationships among the RMSJ of all joint movements suggested a tendency toward a level similar to that of the dominant hand. The results also indicated that decreased ipsilateral brain activity in brain regions related to motor learning reflects the decreased psychological stress for a complex task that required a high level of motor performance skill. A large sample study reported a significant positive correlation between asymmetry of motor performance and hand movement differences in ipsilateral deactivation . The resThis study has some limitations. The execution speed, smoothness of upper extremity joint movement, perceived psychological stress, and brain activity pattern were examined under one condition only. We were unable to determine the extent to which the acquired chopstick operation skill was transferred to various situations during a meal. Moreover, all participants had enough chopstick-use experience with their dominant hands. Thus, the action model of chopstick operation was already stored as memory. Additionally, we set a passive control group that did not experience laboratory training to prove that the change in chopstick operation skills and brain activity deviated from the type of general variations that one would expect from environmental influences. Therefore, we were unable to remove the influence of familiarity with the laboratory equipment and environment due to a laboratory training experience. The fNIRS had a low spatial resolution and could not evaluate the subcortical regions that contributed significantly to motor learning. Finally, this study did not conduct a follow-up assessment post-training. Therefore, we were unable to determine whether the acquired skill was long-lasting.Varied conditions, follow-up assessments, inclusion of active controls, extrinsic feedback, occupational therapy, and neuromodulation methods are recommended in future studies.5The 6-week chopstick training program for the non-dominant hand improved speed and smoothness of chopstick operation with that hand and led to the acquisition of a chopstick performance level similar to that of dominant hand chopstick use. These data may be useful in the rehabilitation of patients requiring a change in handedness due to stroke or other conditions affecting the dominant hand. Psychological and functional brain assessments can be helpful for assessing proficiency in chopstick training skills.


Moore’s Law is approaching its end as transistors are scaled down to tens or few atoms per device, researchers are actively seeking for alternative approaches to leverage more-than-Moore nanoelectronics. Substituting the channel material of a field-effect transistors (FET) with silicene is foreseen as a viable approach for future transistor applications. In this study, we proposed a SPICE-compatible model for p-type (Aluminium) uniformly doped silicene FET for digital switching applications. The performance of the proposed device is benchmarked with various low-dimensional FETs in terms of their on-to-off current ratio, subthreshold swing and drain-induced barrier lowering. The results show that the proposed p-type silicene FET is comparable to most of the selected low-dimensional FET models. With its decent performance, the proposed SPICE-compatible model should be extended to the circuit-level simulation and beyond in future work. In the modern lives, the computing power of digital devices has been improved by the technology innovations in the miniaturisation of semiconductor transistors . The famAmong the options in the more-than-Moore race, two-dimensional (2D) materials have emerged as the prospective contenders owing to their atomically thin structure. Following the success of graphene since 2004 , researcet al. [In 2015, Tao et al.  fabricatet al. –13 and pet al.  forms. H3 monolayer. In addition, a SPICE-compatible model was created to facilitate studies beyond the device-level simulation [Concerning the computational models of silicene-based transistors , 17, rigmulation , such asFig 1 shows the schematic diagrams of the proposed AlSi3 FET and the simplified top-of-the-barrier (ToB) nanotransistor circuit model. In this study, we developed of a SPICE-compatible model for the proposed AlSi3 FET from the ToB nanotransistor model and benchmarked its device performance metrics with other published low-dimensional transistor models. Section 2 describes the modelling procedures to obtain the current-voltage (I-V) characteristics and the respective model evaluation methodology. Section 3 discusses the device performance of AlSi3 FET with respect to its close low-dimensional contenders. Finally, Section 4 includes the conclusion of this work and future work recommendation.Fig 2.This section describes the overall modelling procedures employed in this study, where the overall flowchart is shown in 3 monolayer  was adapted from published DFT study [The atomic structure of the AlSiFT study . By usinFT study , the eleous work .Table 1 summarises the device parameters of the AlSi3 FETs. In the ToB nanotransistor model [P = (PS+PD)−P0 where PS and PD are the non-equilibrium charge densities at the source and drain terminals, respectively, and P0 is the equilibrium charge density. The self-consistent potential USCF at the ToB is obtained by usingq is the constant for electric charge and the total terminal capacitances is expressed as CΣ = CG+CD+CS. Because the source terminal is always set to be zero, αS can be ignored. In an ideal FET, the perfect gate and drain control parameters αG = 1 and αD = 0 [αG = 0.880 and αD = 0.035 [or model , net indd αD = 0  are used = 0.035  were use3 FET can be obtained in terms of VDS and VGS, by employing Landauer-Büttiker ballistic transport equation [g is the degeneracy factor (set as 2 to include up and down spins); ℏ is the Planck’s constant; and kB is the Boltzmann constant. Fig 3(A) shows the I-V characteristics for AlSi3 FET produced by the ToB nanotransistor model.Subsequently, the current-voltage (I-V) characteristics of p-type AlSiequation  along wiequation , given aUSCF, given asPj}{k−j}{ for each respective |VGS|j|VDS|k−j term were computed and optimised using MATLAB curve fitting tool. By using the MATLAB Curve Fitting tool, it was found that the lower orders of polynomial equations fail to fit the USCF well and, as a result, are unable to reproduce accurate I-V characteristics. Therefore, the fifth-order polynomial equation (highest number of order available in the MATLAB Curve Fitting tool) is chosen although the resulting equation is slightly long. The expansion of Eq  and the respective coefficients Pj}{k−j}{ are attached along with the SPICE model library files in the Fig 3(B) shows the I-V characteristics for AlSi3 FET produced by the SPICE model.Following that, the results from ToB nanotransistor were further used to create the SPICE model to allow cross-platform and non-iterative simulation. Moreover, SPICE models are one of the essential tools in the IC design industry for simulation . Before 3 FET. The models were evaluated by using the normalised root-mean-square-deviations (RMSD) [N denotes the total number of data, pi and qi are the values of ith data for the ToB nanotransistor model and the SPICE model, respectively. Fig 4(A) shows the I-V characteristics combining the results of the ToB nanotransistor model and the SPICE model. The point-by-point differences to compute RMSD are plotted in Fig 4(B). Overall, 0.91% of RMSD is produced by the SPICE model when it is benchmarked with the results from the ToB nanotransistor model, indicating that the model has produced a decent fit.In this subsection, a statistical method is employed to evaluate the accuracy of the SPICE model with respect to the ToB nanotransistor model for the proposed AlSis (RMSD) , given a3 FET model. Fig 5 shows the graphical extraction approach to obtain on-to-off current (Ion/Ioff) ratio, subthreshold swing (SS), and drain-induced barrier lowering (DIBL). The proposed AlSi3 FET produces an Ion/Ioff ratio of 2.6×105, a SS of 67.8 mV/dec, and a DIBL of 48.2 mV/V.We can analyse the device performances by extracting the device metrics from the proposed p-type AlSi3 FET with respect to other low-dimensional FETs. We have selected other published works on low-dimensional FETs to fairly assess the device performance of the proposed AlSi3 FET. The selected published models include co-decorated SiNR FET [2) FET [Fig 6. Regarding the Ion/Ioff ratio, the performance of AlSi3 FET model is slightly inferior to 27-ASiNR FET [2 FET [3 FET model is also slightly higher than the 27-ASiNR FET [3 FET model is also outperformed by 27-ASiNR FET [3 FET model is still a prospective alternative for future nanotransistor applications.In this work, we compared our proposed p-type AlSiSiNR FET , 27-ASiNSiNR FET , Si nanoSiNR FET , Si thinSiNR FET , carbon SiNR FET , graphenSiNR FET , black pSiNR FET , and mon [2) FET . To concSiNR FET  and MoS2T [2 FET . RegardiSiNR FET . ConcernSiNR FET  and CNT SiNR FET . HoweverSiNR FET . TherefoIn this paper, we have investigated a SPICE-compatible model for p-type uniformly Al-doped silicene FET. Following that, the device performance of the proposed model is compared with other published low-dimensional nanotransistors. Although the proposed silicene FET is slightly inferior to a few competitors, silicene-based FETs are still one of the potential ways for more-than-Moore nanoelectronic applications owing to its Si-based nature. This work can be extended by performing further circuit-level simulation beyond the transistor device level by using the proposed SPICE model.S1 File(DOCX)Click here for additional data file.


BackgroundDirect-acting antivirals (DAA) have revolutionized the treatment of chronic hepatitis C patients. However, the real-life data regarding its use in a human immunodeficiency virus (HIV) co-infection from a developing country is lacking. We aimed to see the efficacy of DAA in hepatitis C virus (HCV)/HIV co-infected populations.MethodsIn this prospective, observational, intention-to-treat study from Nepal, treatment-naïve patients undergoing treatment for chronic HCV in HIV co-infected individuals with DAA were studied. Patients on nevirapine were switched to efavirenz or atazanavir. Patients received sofosbuvir/ledipasvir or sofosbuvir/daclatasvir with or without ribavirine. Sustained virological response (SVR) at week 12, adverse events, and treatment compliance were evaluated. Treatment efficacy was compared between cirrhotic and non-cirrhotic patients.ResultsOf 218 patients presenting with an anti-HCV report, 181 (83%) had detectable HCV RNA. Eighty-five  patients were having ART at presentation. Three patients could not complete treatment due to gall stone pancreatitis and 82 completed treatment. Twenty-nine  were cirrhotic at presentation. Fifty-one  patients were genotype 3, 27 (33%) were genotype 1, three (4%) were mixed 1a/3, and one (1%) was 6. Seventy-four  had SVR12. Non-cirrhotics had 96% SVR compared to 79% in cirrhotics. SVR in genotype 3 was 88% while it was 93% in genotype 1.ConclusionsReal-life experience showed that the DAAs are equally effective in HCV HIV co-infected patients. In non-cirrhotic patients, the result is comparable to mono-infected patients. Genotype 3 co-infected are also difficult-to-treat patients. DAA treatment is well-tolerated in HCV/HIV co-infected patients, and there was no dropout during treatment. It is estimated by World Health Organization that there are 35 million people living with human immunodeficiency virus (HIV) globally . The samThe potent and efficacious direct antiviral agent (DAA), which is well-tolerated, has drastically changed the prognosis of chronic hepatitis C (CHC) treatment. It is estimated that compared to HIV-negative populations, the risk of HCV infection is six times higher for HIV-positive individuals . The preNepal is among the low prevalence country of HCV-infected individuals. The prevalence is less than 1% in different studies -11. It hThis is a prospective, observational study between April 2015 and July 2017 from Nepal. A public call was made for enrollment for the treatment and a discount on various tests and medicine was announced. Treatment-naive patients with a history of intravenous (IV) drug abuse, presenting with a positive anti-HCV blood test report, who were co-infected with HIV were included in the study. An HCV RNA quantitative test was done by real-time PCR in all individuals. Those with detectable RNA were subjected to a regular blood test, genotyping of HCV RNA, ultrasound examination, and fibroscan  after overnight fasting. Cirrhotic patients on ultrasound or patients with a fibroscan value of more than 12.5 kPa underwent upper gastrointestinal (GI) endoscopy examination. Those with ascites in ultrasound underwent ascitic fluid analysis, including adenosine deaminase (ADA), to rule out spontaneous bacterial peritonitis and tubercular peritonitis. If there was any suspicion of additional etiology of cirrhosis , other investigations , serum immunoglobulin G (S IgG), iron profile, copper profile, etc.) were done.For the treatment of HCV chronic hepatitis, two generic DAAs with or without ribavirine were used. For genotype (GT) 1, a fixed-dose combination of sofosbuvir 400 mg plus ledipasvir 90 mg was given for 12 weeks without ribavirine for chronic hepatitis and compensated LC patients. For decompensated liver cirrhosis (LC) weight-based ribavirine, 1000 mg for < 75 kgs and 1200 mg for > 75 kgs were given for the same period. For GT 3 patients and other GTs, a pan-genotypic combination of sofosbuvir 400 mg and daclatsvir 30 mg or 60 mg (depending upon the types of DAA used) was used for 12 weeks in non-cirrhotic patients and 24 weeks in cirrhotic patients. For cirrhotic patients, weight-based ribavirine, 1000 mg for < 75 kgs and 1200 mg for > 75 kgs was given for 24 weeks duration. The dose of ribavirine was adjusted as per the hemoglobin report. For more than 10 gm/dL, a full dose was given. Two hundred mg/day was reduced if it was between 8.5-10 mg/dL.Patients on nevirapine were switched to efavirenz or atazanavir before the initiation of treatment to avoid drug-drug interaction. Priority was given to change to atazanavir, as the dose of daclatasvir required was only 30 mg OD. The dose of daclatasvir was adjusted accordingly. Sustained virological response at week 12 , adverse events, and treatment compliance were evaluated. Treatment efficacy was compared between cirrhotic and non-cirrhotic patients.The schedule of visits after the initiation of treatment was days 14, 42, and 84. At every visit, the occurrence of adverse events related to the complications of the DAA treatment, antiretroviral (ARV) treatment, and treatment related to liver disease, if any, was collected and reported. Treatment changes were made accordingly as per standard protocol.Data were put in Microsoft Excel  format, and statistical analysis was done using SPSS ver 23 software . Written informed consent was taken from each patient before starting treatment. Institutional review board (IRB) approval was given by the IRB of Norvic International Hospital.Two hundred eighteen presented with a positive anti-HCV positive report during the study period. One-hundred eighty-one 181; 83%) had detectable HCV RNA by polymerase chain reaction (PCR). Spontaneous HCV clearance was evident in 17% of subjects; those who tested positive for anti-HCV antibodies. Eighty-five (85) patients were taking ART at the time of presentation and were included in the study. A total of 21 patients had to change the ART regimen. The ART regimen before starting DAAs is shown in Table 81; 83% hThree patients could not complete treatment due to gall stone pancreatitis and 82 completed treatment and were available for the final analysis.Out of 82 patients treated, 78 patients were male and four patients were female showing male predominance. The majority of the patients were from 20-40 age groups, showing this disease as a disease of younger age group in our country. All but one patient had contracted the disease by sharing needles. One female had contracted both viruses from her husband. Only two male patients gave a history of occasional male sexual partners.The summary of lab parameters is given in Table Patients were grouped into the cirrhotic and non-cirrhotic groups. Spearman correlation between aspartate aminotransferase to platelet ratio index (APRI) and fibroscan score was seen. Spearman's correlation coefficient was positive with an r-value of 0.78 Figure , and thiThere was a predominance of GT 3. Fifty-one 62%) patients were GT 3, 27 (33%) were GT 1, three (4%) were mixed 1/3, and one (1%) was 6  patients had a low viral load while 12% of patients had a very high viral load. The ARV regimen before the initiation of treatment is given in Table A total of 74 (90%) had SVR12. Non-cirrhotics had 96% SVR compared to 79% in cirrhotics. SVR in GT 3 was 88% while it was 93% in GT 1. These results are comparable with the results of treatment of non-cirrhotic mono-infected individuals (data not shown). There was no correlation between viral load and SVR. High fibroscan value and high APRI were associated with no SVR. The mean value was higher in the non-SVR group, and it was statistically significant Tables .The side effects developed during treatments were not life-threatening. There were three deaths during the study. Those deaths were due to complications developed after gall stone pancreatitis. One patient gave a history of binge drinking before admission. Five compensated LC patients developed ascites after one month of treatment. However, they were managed with diuretic therapy and fluid restrictions. Six patients had a fall in hemoglobin after taking ribavirin. It was managed with dose reduction and oral iron therapy. Toward the end of the treatment, all had hemoglobin > 10 mg/dL. Four patients developed jaundice after changes of nevirapine to atazanavir. However, there was no rise in liver enzymes on serial follow-up and the jaundice was predominantly unconjugated hyperbilirubinemia; no intervention was done. The side effects are listed in Table With the availability of highly effective DAAs, global HCV elimination is a burning topic. Will we be able to eradicate HCV from the globe by 2030 is the million-dollar question. HIV/HCV co-infected patients were regarded as difficult to treat patients in the interferon era. However, our study result showed that these patients are no more difficult to treat with the advent of new DAAs. It clearly showed that IV drug abuse is less common among females in the Nepali community. The mode of transmission was the exchange of needles in these patients, making this disease specific to the disease caused by addiction and self-inflicted injury. It was interesting to note that all but two patients had tattoos in different parts of the body, showing a special affinity of the drug abusers’ community towards tattooing. Eighty percent of patients were from the 20-40 years age group, showing that HCV disease is a disease of younger age groups in Nepal. The problem of males having sex with men is less common in Nepal, as only two patients showed this behavior.There was a male preponderance in our study, as only 5% of the total population were females and the rest 95% were males. This is in tune with a previous study . It cleaGenotype 3 is the most prevalent GT in Nepal. It was 59.8% in one study from Nepal . A similThe correlation of APRI, fibrosis, and fibroscan have been studied in monoinfected CHC patients -17. HoweAlthough there were three deaths during the study, those were not directly related to DAA-related adverse events. All three patients were cirrhotic and were diagnosed to have gall stone pancreatitis. Pancreatitis after the initiation of DAAs is not reported so far. However, there have been case reports regarding the development of pancreatitis after taking ribavirine . In our It is now well-documented that HIV infection accelerates HCV-associated liver fibrosis, most notably in those with more advanced immunodeficiency. It results in high rates of end-stage liver disease, and survival is shortened after hepatic decompensation events -20. DespGenotype 3 co-infected are also difficult-to-treat patients. It was also true for HCV/HIV co-infection. SVR in GT 3 was 88% while it was 93% in GT 1. When compared to a similar number of mono-infected individuals in the same institute for the same duration, SVR was less. When we analyzed the data, we found that for fibrosis (as evident by the fibroscan score), the mean score was more in co-infected individuals. More fibrosis at the time of presentation may be the cause of decreased SVR in these patients. GT 3 was also associated with two decompensated cirrhosis patients. Out of the five patients who showed signs of decompensation after the initiation of treatment, four were GT 3. Thus, GT 3 was found to be the more difficult-to-treat group as compared to GT 1.Nepal already presented remarkable achievements in infectious diseases, particularly the HIV response. WHO has set a goal to eliminate HCV by the year 2030 AD. With the availability of generic DAAs and acceptable SVR in mono and co-infected patients, we may attain the goal set by WHO. As the target group is defined (IV drug abusers), we can identify, screen, and treat those individuals.